Molecular Biology Reports最新文献

Introduction: Rice kernel smut, caused by Tilletia horrida, is becoming an increasingly serious disease in hybrid rice planting, leading to production losses and quality decline of male-sterile rice varieties. Successful infection requires an efficient energy source that the pathogen obtains from rice plants, such as carbohydrates. Glycoside hydrolases (GHs), one of the largest sub-families in the cell wall-degrading enzyme family, play a key role in the infection progress of pathogens. To investigate their roles in facilitating infection, in this study, we identified and characterized genes encoding GH family proteins of T. horrida and further explored the functions and structures of these genes.

Materials and methods: Through genome-wide sequencing and bioinformatics analyses, 52 GH genes were identified from T. horrida, named ThGhd_1 to ThGhd_52. The subcellular location, conserved motifs, and structures of ThGhds were identified by bioinformatics analyses.

Results: Phylogenetic analysis revealed that ThGhds with similar domains clustered together, although some proteins clustered in different branches, which might reflect functional diversity. Protein-protein interaction network analysis revealed that ThGhds interact with partner proteins involved in reactive oxygen species signaling, protein kinase activity, and plant hormone signal transduction pathways. RNA-sequencing analysis showed that the expression of ThGhd genes responded differently at different infection time points, with dynamic changes detected during the T. horrida infection process, indicating that these genes are involved in interactions with rice and have potential roles in pathogenic mechanisms.

Conclusions: The results of this study provide valuable resources for the structure elucidation of GH family proteins of T. horrida and can help to further elucidate their roles in pathogenesis.

Background: Excessive chondrocyte death is a critical player in the process of osteoarthritis (OA). The present study was aimed to study the role of receptor-interacting serine/threonine kinase (RIP) 1-mediated signaling for programmed cell death in OA.

Methods: In the present study, RIP1 protein expression was evaluated in mouse OA cartilage and cultured primary murine chondrocytes exposed to tumor necrosis factor-alpha (TNF-α). Protein expression involved in necroptosis and apoptosis and chondrocyte-derived extracellular matrix were examined. Inhibition of RIP1 was conducted using the RNAi technique and pharmacological inhibition. Western blot, immunohistochemistry, and immunofluorescence examination were applied.

Results: The protein presence of RIP1, but not RIP3, was increased in the mouse OA tissue and cultured chondrocytes exposed to TNF-α. Knockdown of RIP1 increased protein expression of collagen II and sex-determining region Y-box transcription factor 9, and reduced protein expression of matrix metallopeptidases 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5. Inhibition of RIP1 reduced the phosphorylated NF-κB signals, decreased cell apoptosis, and restored extracellular matrix expression in cultured chondrocytes. Both RNAi and pharmacological inhibition of RIP1 decelerated the progress of OA in mice.

Conclusion: RIP1 regulates chondrocyte apoptosis through NF-κB signaling. Inhibition of RIP1 provides a novel therapeutic approach for OA therapy.

Background: Previous studies have demonstrated that miR-146a-5p negatively regulated the intrinsic immune and inflammatory responses, whether the miR-146a-5p-enriched exosomes possess the anti-inflammation effect remains unclear. This study aimed to investigate the effect of miR-146a-5p-enriched exosomes on M1 macrophage activation and inflammatory response and the potential molecular mechanism.

Methods: GEO database was used to analyze the expression of miR-146a-5p in serum exosomes of MASH patients. MiR-146a-5p levels in primary hepatocytes, macrophages, and serum exosomes of MASH mice were measured. MiR-146a-5p-enriched exosomes were constructed and the effects on M1 macrophages activation and inflammatory factors release were investigated. The target gene of miR-146a-5p was predicted and verified.

Results: Serum exosomal miR-146a-5p level was decreased in MASH patients analyzed by GEO database. The miR-146a-5p levels in primary cultured hepatocytes and macrophages of MASH mice were decreased. Serum exosomal miR-146a-5p level was decreased and negatively correlated with the concentrations of IL-6 in MASH mice. Furthermore, miR-146a-5p-enriched exosomes inhibited the M1 macrophages activation and the expression of pro-inflammatory factors MCP-1, IL-6, and TNF-α. CD80 was predicted as the potential target gene of miR-146a-5p, and the expression of CD80 was regulated by miR-146a-5p. In addition, the inhibitory effect of miR-146a-5p on M1 macrophages activation and inflammatory factors release was restored when CD80 was over-expressed.

Conclusions: This study demonstrated that miR-146a-5p-enriched exosomes can inhibit the M1 macrophages activation and reduce the release of pro-inflammatory factors by targeting CD80.

Background: A hydrolytic enzyme called protease from microbial sources has expansive applications in leather as well as tannery industries and thus leaves no choice but to discover a potential candidate for efficient yield of protease with eccentric characteristics.

Methods and results: In the current study, using skim milk agar medium, three bacterial strains (Pro SS14, Pro NTL1, Pro SM) were identified for protease production out of thirty tannery waste samples. The bacterial isolates were identified through morphological, biochemical and molecular basis and the highest protease producer (134 U/mL) having the paramount protein content (8.75%) with the greatest biomass (absorbance 1.7 at 600 nm) was further bioinformatically identified as Bacillus subtilis Pro NTL1. Moreover, the cultural conditions-pH, temperature, incubation period and salinity were tested to get the optimized condition for the three isolates where Bacillus subtilis Pro NTL1 gave best growth at pH 9 demonstrating alkaline protease producing capability, at 45 °C for 72 h proving its thermotolerance characteristics and at 3% NaCl concentration declaring its halotolerance property. Furthermore, following Kirby-Bauer agar disc diffusion technique, the antimicrobial susceptibility pattern was investigated showing Bacillus subtilis Pro NTL1 was resistant to ampicillin, penicillin, sulfafurazole and sensitive to amoxicillin, gentamycin, cefotaxime, kanamycin, imipenem, tetracycline. Additionally, Bacillus subtilis Pro NTL1 showed antagonistic activity against three pathogens named Bacillus cereus, Bacillus megaterium and Escherichia coli.

Conclusions: Thus, these findings suggest that Bacillus subtilis Pro NTL1 is a newly isolated strain with unique features which might help to contribute in several biotechnological purposes with significant industrial benefits.

Research has demonstrated the high mortality and morbidity associated with B-Acute lymphoblastic lymphoma (B-ALL). Researchers have developed several therapeutic approaches to combat the disorder. Recently, researchers developed chimeric antigen receptors (CARs)-T cells, which recognize antigens independently of major histocompatibility complexes (MHCs) and activate at a higher level with additional persistence. However, relapsing B-ALL has been reported in several cases. This review article was aimed to collecting recent information regarding the mechanisms used by B-ALL-related lymphocytes to escape from CAR-T cells and the plausible resolution projects.

Background: Macrophages are major effectors in regulating immune response and inflammation. The pro-inflammatory phenotype (M1) is induced by the activation of the Toll-like receptor 4 (TLR4) on the macrophage surface, which recognizes lipopolysaccharide (LPS), a component of Gram-negative bacterial wall, and by the binding of interferon-gamma (IFNγ), a cytokine released by activated T lymphocytes, to its receptor (IFNGR). Among the pathways activated by LPS/IFNγ is the Notch pathway, which promotes the M1 phenotype. Conversely, 17β-estradiol (E2) has been shown to blunt LPS-mediated inflammatory response. While it has been shown that E2 regulates the activity of the Notch1 receptor in human endothelial cells, there is no evidence of estrogen-mediated regulation of Notch1 in macrophages.

Methods and results: In this study, RAW 264.7 cells were stimulated with LPS/IFNγ in the presence or absence of E2 and/or N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of γ-secretase, the enzyme involved in Notch activation. The effects of treatment on inducible nitric oxide synthase (iNOS), on components of the Notch pathway, and MAPK (mitogen-activated protein kinase) were assessed by quantitative PCR and Western blotting. We found that E2, through a mechanism involving the inhibition of p38 phosphorylation, reduces the activation of Notch1 induced by LPS/IFNγ. On the contrary, Notch1 exerts a negative control on the estrogen receptor α (ERα) since Notch1 inhibition increases the protein levels of this receptor.

Conclusion: In conclusion, we report for the first time a Notch-ERα interaction in macrophages. Our data suggest that E2 may reduce LPS/IFNγ-mediated M1 pro-inflammatory phenotype in macrophages by inhibiting Notch1. This finding encourages further studies on Notch1 inhibitors as novel treatments for inflammation-related diseases.

Background: The KCNJ11 gene belongs to the potassium channel gene family. It has a major role in the secretion of insulin. Genetic variations in KCNJ11 are possibly responsible for the progression of type 2 diabetes mellitus (T2DM). In this study, we investigated the possible correlation between KCNJ11 (rs5210) gene polymorphism and T2DM.

Subjects and method: This study included 92 individuals divided into two groups. Group 1 included 46 type 2 diabetic patients. Group 2 (control group) included 46 healthy participants. A complete history was taken and a full physical examination was performed. Anthropometric data were measured. Laboratory investigations included fasting blood glucose (FBG), two hours post-prandial blood glucose (2HPPBG), glycated hemoglobin (HbA1c), and fasting lipid profile. KCNJ11 (rs5210) single nucleotide polymorphism was detected by polymerase chain reaction restriction-fragment length polymorphism (PCR-RFLP).

Results: Both AG and GG genotypes were associated with increased risk for T2DM (OR 5.2, 95% CI 1.32-20.5, P = 0.01 for AG; and OR 18.2, 95% CI 2.99-31.7, P = 0.002 for GG). Also, the frequency of the G allele was significantly higher in type 2 diabetic patients compared to healthy controls (50% versus 23.9%, respectively). The G allele of rs5210 in KCNJ11 contributed to an increased risk of T2DM (OR 3.18, 95% CI 1.31-7.75, P = 0.01). There was a statistically significant association between increased 2HPPBG and HbA1c levels and the carrier of AG and GG genotypes (P = 0.01 and 0.007, respectively). There was a statistically significant association between total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-c), and high-density lipoprotein-cholesterol (HDL-c) levels and the carrier of AG and GG genotypes (P < 0.001, 0.02, and 0.007, respectively). Regression analysis detected that body mass index (BMI), 2HPPBG, TC, triglycerides (TG), and the G allele of rs5210 in KCNJ11 gene showed a significant association with T2DM (P = 0.004, 0.042, 0.003, 0.006, and 0.01, respectively) while no association was observed with FBG, HbA1c, LDL-c or HDL-c (P = 0.099, 0.123, 0.522, and 0.765, respectively).

Conclusion: KCNJ11 rs5210 genetic polymorphism may raise the risk for the occurrence of T2DM among Egyptians.

Background: Developing a molecular signature associated with CD4 + T cell infiltration is essential for identifying biomarkers in abdominal aortic aneurysms (AAA). Establishing such a signature is vital for improving diagnostic accuracy and therapeutic strategies for AAA. This study focuses on CD4 + T cells, which are pivotal in the immune microenvironment of AAA, to pinpoint key targets.

Methods and results: We identified CD4 + T cell-related biomarkers in AAA using bulk and single-cell RNA sequencing data from the GEO database. We employed CIBERSORT to assess immune cell infiltration and applied weighted gene co-expression network analysis and differential expression analyses to pinpoint key genes. A nomogram and related score were developed based on these genes. Single-cell RNA sequencing further analyzed their expression across cell types, and KEGG/GO analyses were conducted for candidate genes. Four genes (NUPR1, CCL4L2, CCL3L3, MMP9) were identified finally. Validation via qPCR and immunohistochemistry showed NUPR1 downregulation in aneurysms and an inverse relationship with CD4 + T cells infiltration. Immunofluorescence results indicated NUPR1 was mainly expressed in the cytoplasm of vascular smooth muscle cells (VSMCs). After VSMCs-specific overexpression of NUPR1 via adeno-associated virus, the AAA diameter decreased, while treatment with the NUPR1 nuclear translocation inhibitor ZZW-115 hydrochloride had no effect on AAA size. Overexpression of NUPR1 in VSMCs suppresses the migration of CD4 + T cells.

Conclusion: The four-gene signature accurately predicts CD4 + T cell infiltration in AAA patients and may serve as a clinical index. NUPR1 could be a therapeutic target for the interaction between CD4 + T cells and AAA.

Background: Adamantinomatous craniopharyngiomas (ACPs), commonly seen in pediatrics and adults often present with large cystic cavities that can compress surrounding tissues, causing severe visual and endocrine symptoms. Complete resection of cystic ACP is challenging, frequently leading to postoperative recurrence. The composition of the cystic fluid is complex, and to date, there has been limited research focusing on exosomes within ACP cyst fluid.

Methods: We collected cyst fluid from 12 ACP patients and confirmed the presence of exosomes. Subsequently, we conducted exosomal proteomic analysis using LC-MS/MS. The patients were divided into pediatric and adult groups for the analysis of differential protein enrichment, followed by comprehensive bioinformatics analysis, including GO analysis, KEGG analysis, and PPI network analysis, among other functional pathway and protein interaction analyses. Immunohistochemistry was used to determine the tissue expression distribution of the differential protein APOA1.

Results: In our data analysis, 64 significantly differentially expressed proteins were identified, with 37 being overexpressed in the pediatric group and 27 in the adult group. Our results revealed that exosomal proteins in the pediatric group were predominantly enriched in modules and pathways related to high-density lipoprotein particle, apolipoprotein receptor binding, and the PPAR signaling pathway. Additionally, APOA1, as the hub protein with the highest connectivity in the differential protein interaction network, may play a critical role in β-amyloid metabolism pathways in pediatric ACP.

Conclusion: This study is the first to construct a proteomic map of ACP cyst fluid exosomes, suggesting significant differences in the tumor microenvironment's lipid metabolism between pediatrics and adults.